High-Throughput Screening Assay Development

Vitamin B6 is one of those things that most people are probably dimly aware of and likely couldn’t tell you what it does. In fact, B6 is involved in a whole host of processes and is essential for things as diverse as brain development, blood circulation, proper immune function, and improved mood. In total, B6 is known to be involved in around 200 reactions and processes in your body.  The problem with B6 (as with a lot of micronutrients) is that many people are not getting enough, the issue for B6 is compounded by the fact that the currently available B6 screening assays are lengthy and complex, making it challenging for physicians to ascertain or monitor a patient’s B6 levels, making it hard to diagnose a deficiency.

The need for a better clinical assay

Current B6 screening assays require extensive sample preparation and lengthy analysis times. The development of a rapid, high-throughput assay is an unmet need for front-line clinicians. In a recent publication by scientists at ARUP Laboratories in Salt Lake City, the authors describe the development of a new high-throughput assay capable of screening for the circulating form of vitamin B6 in the plasma of adults and children.^[1] A clinically relevant assay would need to be simple and efficient enough to routinely monitor B6 concentrations in the general population, sensitive enough to detect any low-level variations across populations, and reliable enough to function as a front-line method for the identification of vitamin B6 deficiency.

Screening assay development strategy

To develop a suitable high throughput assay, the researchers mapped each step in the existing analysis process with the goal of streamlining both the complex and time-consuming sample preparation portion of the protocol, and the lengthy instrument analysis segment. Any changes made to the assay to shorten and simplify would also need to have no impact on assay performance, leaving the final protocol at least as robust and sensitive as the conventional vitamin B6 detection assay it was intended to replace.

Plasma samples from both adults and children (aged 1-17) obtained as residual aliquots from routine lab testing were used for method comparison and internal standard calibration studies.  The authors developed a simplified sample preparation protocol using AcroPrepÔ Advance 96-well filtration plates. Following the addition of internal standards and TCA, plasma samples were vortexed, then centrifuged to clear any particulate matter. The supernatants were then filtered using the AcroPrep filtration plates.

Filtration plates increase speed and throughput

The use of filtration plates is a simple strategy that can be employed to accelerate many different lab protocols where filtration is required. The 96-well filter plates enable more samples to be filtered in one go, are automation-friendly, and amenable to filtration using either centrifugation or standard lab vacuum manifolds.

Pall’s AcroPrep plates are designed in accordance with the ANSI/SBS X-2004 standards and are available in 24, 96, and 384 well formats to suit different application needs.

Filtered plasma samples were set up in an automated workstation for LC-MS/MS analysis. While current B6 analysis methods use a single column for sample separation, the ARUP scientists elected to use an Alternating Column Regeneration (ARC) platform.  An automated dual-column system that yields a 30% reduction in analysis time. The choice of LC-MS/MS analysis enables a more rapid workflow with greater sensitivity and specificity when compared to the traditional B6 assay’s use of HPLC coupled to a fluorescence detector.

Exploring vitamin B6 levels in a cross-section of the population

In evaluation studies of the new and old assay methods, the authors found no carryover, loss of sensitivity, or specificity. The new assay remained linear down to 500 nmol/L, which is well above the sensitivity level required for physiological relevance.

After validating the assay, the scientists put it to use analyzing sample population demographics. They found higher circulating vitamin B6 in men compared to women, the lowest rates of B6 deficiency in children and the highest in elderly adults. The team also demonstrated the presence of significant seasonal variance with circulating B6 lower during winter and spring months, and higher during summer and fall.

This research represents a promising new method for front-line clinical evaluation of circulating vitamin B6 levels in patients. Combining simplified, automated sample preparation with high-throughput LC-MS/MS to significantly shorten workflow and instrument analysis time. This new assay will allow for a broader assessment of Vitamin B6 in the general population and hopefully lead to a better understanding of the true prevalence and effects of vitamin B6 deficiency.

The AcroPrep Advance Filter plates are an excellent tool for increasing throughput, speed, and enabling the automation of a multitude of lab protocols and a wide range of analytical applications. You can learn more about the AcroPrep Advance 96-well filter plates used in this study on the Pall website (for research only).